Utility of Enzyme Crosslinked Matrices
Enzyme crosslinked matrices are formed in a variety of applications in the food, cosmetic, and medical industries. In medical applications in particular, enzyme crosslinked hydrogels are widely used in a variety of medical applications including tissue sealants and adhesives, haemostatic preparations, matrices for tissue engineering or platforms for drug delivery. While some hydrogels such as gelatin and poloxamer may be formed as a result of physical interactions between the polymer chains under specific conditions, e.g change in temperature, most polymer solutions must be crosslinked in order to form hydrogels. In addition to the actual formation of the solid gel, implantable hydrogels must be resistant to the conditions that are prevalent in the tissue where they are applied, such as mechanical stress, temperature increase, and enzymatic and chemical degradation. For this reason, in many cases it is necessary to crosslink the hydrogel matrices. The crosslinking may be done outside the body by pre-casting or molding of hydrogels. This application is used mainly for tissue engineering or drug delivery applications. Alternatively, crosslinking may be done inside the body (in situ gelation or crosslinking) where a liquid solution is injected or applied to the desired site and is cross linked to form a gel.
Gel formation can be initiated by a variety of crosslinking approaches. Chemical approaches to gel formation include the initiation of polymerization either by contact, as in cyanoacrylates, or external stimuli such as photo-initiation. Also, gel formation can be achieved by chemically crosslinking pre-formed polymers using either low molecular weight crosslinkers such as glutaraldehyde or carbodiimide (Otani Y, Tabata Y, Ikada Y. Ann Thorac Surg 1999, 67, 922-6. Sung H W, Huang D M, Chang W H, Huang R N, Hsu J C. J Biomed Mater Res 1999, 46, 520-30. Otani, Y.; Tabata, Y.; Ikada, Y. Biomaterials 1998, 19, 2167-73. Lim, D. W.; Nettles, D. L.; Setton, L. A.; Chilkoti, A. Biomacromolecules 2008, 9, 222-30.), or activated substituents on the polymer (Iwata, H.; Matsuda, S.; Mitsuhashi, K.; Itoh, E.; Ikada, Y. Biomaterials 1998, 19, 1869-76).
However, chemical crosslinking can be problematic in food, cosmetic, or medical applications because the cross-linkers are often toxic, carcinogenic, or irritants. Furthermore, they are small molecules that can readily diffuse out of the crosslinked matrix and might cause local or systemic damage.
An alternative to chemical crosslinking is the enzymatic crosslinking approach. These approaches to initiate gel formation have been investigated based on a variety of different crosslinking enzymes. Examples include enzymatic crosslinking of adhesives, such as mussel glue (Strausberg RL, Link RP. Trends Biotechnol 1990, 8, 53-7), or the enzymatic crosslinking of blood coagulation, as in fibrin sealants (Jackson MR. Am J Surg 2001, 182, 1S-7S. Spotnitz W D. Am J Surg 2001, 182, 8S-14S Buchta C, Hedrich H C, Macher M, Hocker P, Redl H. Biomaterials 2005, 26, 6233-41.27-30).
Cross-linking of a mussel glue was initiated by the enzymatic conversion of phenolic (i.e., dopa) residues of the adhesive protein into reactive quinone residues that can undergo subsequent inter-protein crosslinking reactions (Burzio L A, Waite J H. Biochemistry 2000, 39, 11147-53. McDowell L M, Burzio L A, Waite J H, Schaefer J J. Biol Chem 1999, 274,20293-5). The enzymes which have been employed in this class of sealants are tyrosinase on one hand and laccase and peroxidase on the other hand which acts by forming quinones and free radicals, respectively from tyrosine and other phenolic compounds. These in turn can crosslink to free amines on proteins or to similarly modified phenolic groups on proteins and polysaccharides.
A second cross-linking operation that has served as a technological model is the transglutaminase-catalyzed reactions that occur during blood coagulation (Ehrbar M, Rizzi S C, Hlushchuk R, Djonov V, Zisch A H, Hubbell J A, Weber F E, Lutolf M P. Biomaterials 2007, 28, 3856-66). Biomimetic approaches for in situ gel formation have investigated the use of Factor XIIIa or other tissue transglutaminases (Sperinde J, Griffith L. Macromolecules 2000, 33, 5476-5480. Sanborn T J, Messersmith P B, Barron A E. Biomaterials 2002, 23, 2703-10).
An additional in situ crosslinked gel formation of particular interest is the crosslinking of gelatin by a calcium independent microbial transglutaminase (mTG). mTG catalyzes an analogous crosslinking reaction as Factor XIIIa but the microbial enzyme requires neither thrombin nor calcium for activity. Initial studies with mTG were targeted to applications in the food industry (Babin H, Dickinson E. Food Hydrocolloids 2001, 15, 271-276. Motoki M, Seguro K. Trends in Food Science & Technology 1998, 9, 204-210.), while later studies considered potential medical applications. Previous in vitro studies have shown that mTG can crosslink gelatin to form a gel within minutes, the gelatin-mTG adhesive can bond with moist or wet tissue, and the adhesive strength is comparable to, or better than, fibrin-based sealants (Chen T H, Payne G F, et al. Biomaterials 2003, 24, 2831-2841. McDermott M K, Payne G F, et al. Biomacromolecules 2004, 5, 1270-1279. Chen T, Payne G F, et al. J Biomed Mater Res B Appl Biomater 2006, 77, 416-22.). The use of gelatin and mTG as a medical adhesive is described in PCT WO/2008/076407.
One of the disadvantages of using enzymes as the cross-linkers in crosslinked matrix formation is that they may continue the crosslinking reaction after the desired gel state has been formed. This is often not desired because excessive crosslinking may result in a stiffer, more brittle, and less flexible gel. In addition, the mechanical properties of the crosslinked matrix will continue to change during the lifetime of the gel, making consistent properties difficult to achieve. The continued enzymatic crosslinking beyond the desired crosslinking density results from the ability of the enzyme to continue to catalyze the crosslinking reaction even once a crosslinked matrix or hydrogel has been formed. This depends on the ability of the enzyme to continue to diffuse throughout the matrix even as solution viscosity increases greatly. This view is consistent with Hu et al (Hu B H, Messersmith PB. J. Am. Chem. Soc., 2003, 125 (47), pp 14298-14299) who suggested, based on work done with peptide-grafted synthetic polymer solutions, that during incipient network formation resulting from partial cross-linking of a polymer solution, the solution viscosity rapidly increases while the mobility of the transglutaminase rapidly decreases.
The problem of excessive enzymatic crosslinking leading to a reduction in mechanical properties has been previously documented on several occasions:
Bauer et al. demonstrated that high levels of microbial transglutaminase (mTG) caused excessive cross-linking of wheat gluten proteins leading to a loss of elasticity and mechanical damage of the gluten networks. (Bauer N, Koehler P, Wieser H, and Schieberle P. Studies on Effects of Microbial Transglutaminase on Gluten Proteins of Wheat II Rheological Properties. Cereal Chem. 80(6):787-790).
Sakai et al. found that a larger quantity of covalent cross-linking between phenols was effective for enhancement of the mechanical stability, however, further cross-linking between the phenols resulted in the formation of a brittle gel. (Sakai S, Kawakami K. Synthesis and characterization of both ionically and enzymatically crosslinkable alginate, Acta Biomater 3 (2007), pp. 495-501)
In the case of cofactor-dependent crosslinking enzymes, such as calcium-dependent transglutaminase, removing the cofactor, by binding or otherwise, after a certain reaction time can limit the degree of crosslinking. However, cofactor removal is frequently not technically feasible in hydrogel formation where the hydrogel may trap the cofactor. When using cofactor-independent enzymes, such as transglutaminases available from microbial origin, limited degrees of crosslinking can be obtained by heat treatment of the reaction system. However, such a treatment induces negative side effects on protein functionality and is therefore undesirable to apply. In addition, not all reaction systems are suitable to undergo heat treatment.
Other than resulting in excessive crosslinking within the crosslinked matrix, continued diffusion of the crosslinked enzyme in the matrix after the desired crosslinked state has been achieved also can result in a high rate of enzyme diffusion out of the gel, also known as enzyme elution. This can also be problematic as high levels of crosslinking enzyme released into the body can interact with body tissues and cause local or systemic damage.